Kisiel, John B.

Abstract Aberrant DNA methylation and copy-number alterations (CNA) drive Barrett’s esophagus progression to esophageal adenocarcinoma; however, their combined utility for early detection is unclear. We aimed to identify and validate methylated DNA markers (MDM) and CNAs to distinguish esophageal adenocarcinoma/high-grade dysplasia (HGD) from nondysplastic Barrett’s esophagus (NDBE). In this multiphase, multicenter study, we discovered and validated MDMs and quantified CNAs utilizing whole-genome methylation sequencing of esophageal brushings. DNA biomarkers identified from discovery were further validated in independent patients with paired esophageal brushing and swallowed capsule sponge samples. MDMs were filtered against a reduced representation bisulfite sequencing dataset obtained from independent tissue samples to advance only concordant candidates. CNA burden was quantified using ichorCNA-derived aneuploidy scores (AS). Two hundred MDMs discovered in HGD (N = 18) and esophageal adenocarcinoma (N = 18) versus NDBE brushing samples (N = 18) were tested in independent samples (N = 146). A 52-MDM panel achieved a cross-validated AUC of 0.88 [95% confidence interval (CI), 0.82–0.95]; the addition of AS improved discrimination of HGD/esophageal adenocarcinoma from NDBE to 0.91 (95% CI, 0.86–0.97) AUC. At 80% specificity, the combined model detected 93% of esophageal adenocarcinoma and 88% of HGD cases. In paired capsule sponge samples, a 58-MDM panel achieved a cross-validated AUC of 0.77 (95% CI, 0.66–0.88); a combined 58-MDM and AS model achieved AUC 0.80 (95% CI, 0.7–0.9). MDMs and AS discerned HGD/esophageal adenocarcinoma from normal esophagus/NDBE in endoscopic brushing and capsule sponge samples. This approach may improve Barrett’s esophagus surveillance. Prevention Relevance: This study demonstrates that combining epigenetic and genomic biomarkers across minimally invasive sampling methods can accurately distinguish HGD/esophageal adenocarcinoma from nondysplastic Barrett’s esophagus, offering promising, less invasive strategies to improve BE surveillance and enable endoscopic therapy for esophageal adenocarcinoma prevention and treatment.

Abstract Background Recommended noninvasive strategies for average-risk colorectal cancer (CRC) screening include multitarget stool DNA and fecal immunochemical test from ages 45 to 75 years. With new clinical trials, performance data for next-generation multitarget stool DNA, multitarget stool RNA, and blood-based screening tests are now available. This decision analytical model study evaluated the estimated benefit-to-burden ratio by means of efficient frontiers for noninvasive established and emerging CRC screening strategies. Methods Outcomes were estimated using the Colorectal Cancer and Adenoma Incidence and Mortality microsimulation model for average-risk individuals in the United States. Screening strategies were next-generation multitarget stool DNA (an updated marker panel), fecal immunochemical tests, multitarget stool RNA, or blood-based tests every 1-3 years, over various age ranges. Test performance inputs were derived from recent large clinical trials. A strategy was deemed efficient if no other strategy provided more life-years gained with equivalent or fewer lifetime colonoscopies and near-efficient if within 3 days of life-years gained of the efficient frontier. Results All modeled screening strategies resulted in life-years gained vs no screening. No strategy using blood-based tests was efficient or near-efficient. Overall, 10 strategies were efficient (6 next-generation multitarget stool DNA and 4 fecal immunochemical tests), including 2 strategies among those ages 45-75 years (biennial and triennial next-generation multitarget stool DNA). Overall, 22 strategies were near-efficient, including 4 strategies among those ages 45-75 years (annual, biennial, or triennial fecal immunochemical test; annual next-generation multitarget stool DNA). Conclusion Based on this modeling study, next-generation multitarget stool DNA was the only noninvasive screening test at guideline-endorsed interval and age-recommended ranges that was deemed efficient. Blood-based and multitarget stool RNA strategies were deemed not efficient for primary screening.

Abstract Background: Alaska Native people are committed to increasing colorectal cancer (CRC) screening to reduce the high rate of CRC in this population. We conducted a community-level cluster randomized controlled trial (RCT) to evaluate the impact of multi-target stool DNA test introduction (mt-sDNA; Cologuard® Exact Sciences, Madison WI) on CRC screening in southwest Alaska Native communities using a mt-sDNA pre-processing lab in Anchorage, Alaska. Methods: A total of 32 rural/remote Alaska Native communities were evenly randomized to either high intensity patient navigation or medium intensity mailed health education compared to 14 communities receiving standard of care (screening recommendations at health care visits). The intervention was conducted from April 2021-February 2023. Outcome measures included CRC screening completion within 12 months and method used (mt-sDNA vs colonoscopy). Results: A total of 2032 Alaska Native participants (mean [SD] age, 56 [8.4] years; 817 women [40%]) were enrolled: 587 participants randomized to usual care, 696 participants randomized to the mailed education arm, and 749 participants randomized to the patient navigation arm. The mt-sDNA test was significantly preferred over colonoscopy by both mailed education participants (68%) and patient navigation participants (71%) (p<0.01). Mt-sDNA was somewhat more preferred by older participants ages 65-75 (79%) vs younger participants ages 45-54 (64%). Both men (71%) and women (68%) significantly preferred mt-sDNA compared with colonoscopy (p<0.01). Overall, 38% of those in the patient navigation intervention completed screening compared with 33% in the mailed education intervention and 10% in the usual care arm. No significant differences were observed in mt-sDNA completion between the patient navigation arm (154 participants [40%]) and the mailed education arm (100 participants [36%]) (p= 0.34). Colonoscopy completion (not including follow-up colonoscopies after abnormal stool tests) was higher in both the patient navigation arm (112 participants [44%]) and the mailed education arm (87 participants [43%]) compared with usual care (38 participants [14%]). Over 61% of kits sent to participants were completed and returned, however, 38% of the kits were not able to be processed, primarily because of mail transit time exceeding the manufacturer’s collection-to-processing limitation of 72 hrs. Conclusions: In this RCT among Alaska Native peoples who were not up to date with CRC screening recommendations, both a mailed education intervention and a patient navigator facilitated intervention were effective at increasing CRC screening compared with usual care. Participants overwhelmingly preferred mt-sDNA for screening but limitations in getting samples to the lab in time for analysis meant that about one-third of mt-sDNA samples were unable to be processed. These findings show that screening outreach and mt-sDNA use can substantially increase CRC screening, but shipping and weather issues remain to be addressed for expanding use of mt-sDNA in rural/remote Alaskan communities. Citation Format: Diana Redwood, Christie Flanagan, Lauren Jeffries, Peter Holck, Danika Bailie, Christine Desnoyers, Joseph Klejka, Lila Rutten, John Kisiel. Increase in colorectal cancer screening among rural and remote Alaska Native peoples using the multi-target stool DNA: A randomized controlled trial [abstract]. In: Proceedings of the 18th AACR Conference on the Science of Cancer Health Disparities; 2025 Sep 18-21; Baltimore, MD. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2025;34(9 Suppl):Abstract nr A148.

Background/Objectives: Human endogenous retroviruses (ERVs) are genomic sequences integrated into the human genome from ancestral exogenous retroviruses and are epigenetically silenced under normal conditions. Growing evidence has shown that they can be reactivated in human diseases such as cancers and autoimmune diseases. However, their clinical implications in colon cancer are yet to be explored. Methods: RNA-seq data were downloaded from RNA Atlas and TCGA for cell lines and tissue samples, respectively. After alignment, ERV expression was quantified against comprehensively compiled ERVs (3220). ERV expression profiles were compared between sequencing protocols, cancer and normal cells, and matched tumor and normal tissue pairs. Unsupervised clustering was used to identify ERV-defined tumor subtypes and their associations with clinical and other molecular features. ERV association with disease-specific survival (DSS) was performed using the Cox regression model. Results: PolyA and total RNA protocols were comparable in ERV expression detection. Cancer cells had significantly increased ERV expression and reactivation. Upregulated ERVs were significantly enriched in viral protein interactions with cytokine and cytokine receptors. ERV expression-defined tumor classes were significantly associated with tumor mutation burden and immuno-phenotypes such as antigen processing and presenting machinery and tumor immune infiltration score. Survival analysis identified 152 ERVs to be independently associated with DSS. Conclusions: ERV abnormal expression is common in colon cancer. The ERV-defined subtypes are associated with tumor immunity, and some ERVs are independently associated with patient outcomes.

Abstract Purpose We examined barriers and facilitators to colorectal cancer (CRC) screening among Alaska Native individuals who had never been screened or were not up to date with screening guidelines. Methods As part of a larger study investigating the use of the multitarget stool DNA test in rural Alaska communities, we conducted focus groups and in‐depth interviews with 28 never screened or not up‐to‐date Alaska Native people in two remote communities between November 2022 and July 2023. Participants shared their barriers to CRC screening and offered suggestions to improve programs to better reach those who are guideline discordant. Findings General screening barriers included lack of knowledge, fear of discovering you have cancer, and cultural health beliefs. Colonoscopy‐specific barriers included embarrassment from knowing the local medical staff, having rectal area viewed or touched, fear of pain and injury, difficulty scheduling a procedure due to traditional food gathering activities, lack of provider referrals/reminders, and the high cost of air travel required to get to a colonoscopy facility. Stool DNA‐specific concerns included not feeling that it was as good as colonoscopy, lack of privacy to do the test, and hesitancy collecting stool samples. Suggestions for increasing screening rates included increasing access via paid air transportation, using local indigenous languages, improving provider relationships and reminder systems, and providing CRC and screening education using trusted messengers. Conclusions This was the first exploration of the perceptions of CRC screening among guideline discordant Alaska Native people. Identified themes can be used to improve screening program outreach effectiveness in the future.

6057 Background: The incidence rate of human papillomavirus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) has persistently increased over the past decade and is anticipated to rise further over the next decade. Cost effective, accurate, and less invasive screening options for HPV(+)OPSCC have remained stagnant leading to the interrogation of circulating tumor DNA in liquid biopsies such as plasma. We hypothesized that candidate methylated DNA markers (MDMs), previously described by our group, would be present in the plasma of patients with HPV(+)OPSCC and absent in controls. Methods: HPV(+)OPSCC cases were enrolled from a high-volume referral practice. Cancer-free control patients were identified from the surrounding 7-county catchment area and frequency matched to cases by age and alcohol use. cfDNA was extracted from 4 mL of plasma and bisulfite converted prior to Target Enrichment Long-probe Quantitative Amplified Signal assays for 15 MDMs. Strand counts for individual MDMs were normalized to a methylated genomic reference marker. An MDM score was derived via random forest modeling of the 15 MDMs. Area under the receiver operator characteristic curve (AUC) for discriminating cases from controls was evaluated for individual markers and for the MDM score. The sensitivity of the MDM score at 95% specificity was also evaluated. Results: The study consisted of 96 HPV(+)OPSCC and 100 cancer-free controls. The study population was predominately white (95%) with a median age of 61 yrs (IQR: 52-69 yrs). Eighty-nine percent of the cases were male with cancers affecting either the tonsil in 50 (52%) or base of the tongue in 46 (48%) patients; by design 50% of controls were male. Stage I:II:III:IV disease was present in 66%:22%:9%:3% of the cases. The median (IQR) AUC across the 15 MDMs was 0.83 (0.82-0.85) overall: 0.78 (0.77-0.83) for stage I disease and 0.92 (0.91-0.93) for stage II-IV disease. The MDM score for the combined panel of 15 MDMs achieved a cross-validated AUC of 0.93 (CI: 0.89-0.97) overall: 0.90 (CI: 0.84-0.96) for stage I disease and 0.98 (CI: 0.97-1.00) for stage II-IV disease. At 95% specificity, the cross-validated sensitivity of the MDM score for HPV(+)OPSCC was 80% (CI: 71-87%) overall: 73% (CI: 60-83%) for stage I disease and 94% (CI: 78-99%) for stage II-IV disease. Conclusions: We validated a set of previously discovered MDM markers in HPV(+)OPSCC in a robust case control cohort using plasma. The MDMs were detected in both early and late-stage disease. Additional prospective studies in larger intended use cohorts are needed to validate our results for clinical use.

e17617 Background: Abnormal uterine bleeding (AUB) is a cardinal symptom of endometrial cancer (EC). However, 90-95% of those with AUB will undergo invasive endometrial sampling with the finding of benign etiology. Our group has identified EC-related methylated DNA markers (MDMs) detectable in vaginal fluid obtained via self-collected tampon with high sensitivity to detect EC. To provide alternative vaginal fluid collection options, we compared total DNA recovery and detection of EC MDMs between tampon and swab collection methods. Methods: Patients ≥45 yrs with AUB or undergoing routine gynecologic care, and patients ≥18 yrs with biopsy-confirmed EC or atypical endometrial hyperplasia (AEH) were prospectively enrolled for paired vaginal fluid biospecimens collection. Participants self-collected vaginal fluid via tampon before provider-collected vaginal fluid sampling via swab immediately prior to clinically indicated endometrial sampling or hysterectomy. Cases (EC/AEH) and benign controls were determined by expert review. DNA was extracted from vaginal fluid. Bisulfite converted DNA was then used in long-probe quantitative amplified signal (LQAS) assays for 10 MDMs and 2 reference markers (RMs). Total DNA recovery using the RMs and detection of EC-specific MDMs were compared across sampling techniques. MDMs were standardized using the log of the ratio of the MDM and RM1. Standardized MDMs were compared between swabs and tampons using median and interquartile range (IQR). Results: A total of 40 eligible participants were evaluated in this study, 20 cases and 20 controls. Median time between tampon and swab collection was 27 hours (range 1-53). DNA quantity as measured using both RM1 and RM2 were comparable between swab and tampon in both cases and controls; MDM detection was also comparable between swab and tampon in both cases and controls (Table 1; 8 representative MDMs shown). Though not powered for sensitivity and specificity estimation, EC-related MDMs appear higher in EC cases vs controls, using either sample type. Conclusions: Sampling vaginal fluid with provider-collected swabs or self-collected tampons are both effective for detection of validated EC MDMs. Studies are ongoing to optimize an MDM panel to detect EC in vaginal fluid. Control Tampon Control Swab Case Tampon Case Swab RM1 3.5 [3.3-3.8] 4.2 [3.8-4.2] 3.6 [3.2-3.9] 4.2 [3.8-4.5] MDM1 0.5 [0.4-0.7] 0.4 [0.2-0.6] 1.3 [0.8-1.8] 1.1 [0.6-1.7] MDM2 1.5 [1.2-1.9] 0.9 [0.7-1.2] 3.2 [2.2-4.7] 2.9 [1.7-4.5] MDM3 0.5 [0.4-0.6] 0.4 [0.3-0.5] 1.5 [0.9-2.3] 1.0 [0.8-2.3] MDM4 0.9 [0.6-1.3] 0.5 [0.4-0.7] 2.3 [1.3-3.7] 1.9 [1.0-3.6] MDM5 1.3 [0.6-1.4] 0.8 [0.6-1.0] 3.3 [2.5-4.1] 3.1 [2.4-3.9] MDM6 1.6 [1.3-2.1] 1.2 [0.8-1.7] 3.3 [2.6-4.3] 3.1 [2.3-4.1] MDM7 1.5 [1.1-1.7] 1.0 [0.4-1.4] 3.5 [2.6-4.1] 3.3 [2.1-3.8] MDM8 0.5 [0.3-0.6] 0.5 [0.4-0.7] 1.8 [1.0-2.7] 1.7 [1.0-2.5] Median (IQR) for RM1 and 8 MDMs by case/control and sampling technique.

Background Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the second leading cause of cancer-related deaths in the United States. Screening can prevent CRC by detecting advanced precancerous lesions. Adherence to screening is crucial in reducing CRC disease burden; however, there is limited research on the impact of digital outreach screening uptake and adherence. Objective This study evaluated the impact of different digital outreach channels on patient adherence to CRC screening with a multi-target stool DNA (mt-sDNA) test in a real-world setting. Methods Patients were individuals aged 45 to 85 years with a valid mt-sDNA test order from Exact Sciences Laboratories, LLC (Jan. 1, 2023–Sept. 23, 2023). All patients received letters and phone calls; some received short message service (SMS), email, or both. Adherence and time to test return were compared across digital outreach categories stratified by patient characteristics. Multivariable regression evaluated the association of digital outreach methods with adherence and time to test return. Results Among 2 425 308 patients (43.5% between 50 and 64 years, 58.2% female), digital SMS only (62.7%) was the most common outreach method. Overall adherence was 70.1%, with highest adherence in the digital SMS-plus-email group (72.9%). Mean time to test return from shipment of mt-sDNA kit to receipt of valid test was 25.8 days. In adjusted analyses, patients receiving digital SMS plus email had the highest odds of test return (odds ratio, 1.75; 95% confidence interval [CI], 1.73-1.78; P <.001) and had return times 8.7% shorter than the no-digital-outreach group (95% CI, 8.2-9.2; P <.001). Discussion Among nationally insured individuals within the recommended age range for CRC screening, overall adherence to the mt-sDNA test was in the 70s, with the highest rates in the digital (SMS and email) outreach group and the lowest in the no-digital-outreach group. Conclusions These findings highlight the importance of multichannel navigation in facilitating completion of CRC screening with the mt-sDNA test.

Abstract The substantial carbon footprint imparted by medical services warrants increased attention to their environmental impact. National guideline organizations such as the US Preventive Services Task Force (USPSTF) recommend multiple modalities for average-risk colorectal cancer (CRC) screening with varying resource intensity. The aim of this study was to quantify the environmental burden for 2 of the most used CRC screening modalities, colonoscopy and the multi-target stool DNA (mt-sDNA) test. A validated CRC microsimulation model was used to estimate the number of screening and follow-up tests for a cohort of 1 million average-risk individuals who underwent screening between ages 45 and 75. Component resources used for mt-sDNA, including waste products, energy, and transportation for colonoscopy and mt-sDNA, were collected from January 1, 2023, to January 1, 2024, and converted to carbon-equivalent emissions. Resources used for colonoscopy were captured from the literature. Resources devoted to screening colonoscopy were substantially (59%) higher than those to mt-sDNA, even when including follow-up colonoscopy. Of note, follow-up colonoscopy accounted for the majority (64%) of total emissions for the mt-sDNA screening strategy. Compared with colonoscopy screening, mt-sDNA substantially reduces the carbon emissions attributable to population-level CRC screening. Environmental impact should be included as a factor when choosing among guideline-recommended CRC screening strategies.